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Journal: یاخته
Year:2010 | Volume: | Issue:
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Title

INVITED SPEAKERS; BONE MARROW CELL THERAPY FOR TREATMENT OF HEMOPHILIA A IN MICE

Writers

MUKHOPADHYAY A.

Pages

 Start Page 9 | End Page 10

Abstract

 Objective: Bone marrow (BM) cells can directly differentiate into HEPATOCYTES due to cellular plasticity. Regeneration of nonhematopoietic tissue by BM cells has been proposed as a promising novel class of therapies for the treatment of many diseases and injuries in mammals. HEMOPHILIA A (HA) is an X-chromosomelinked recessive bleeding disorder. Since liver is the primary site of FVIII synthesis, we hypothesized that the partial replacement of mutated liver cells by healthy cells in HA mice could manage the severity of the bleeding disorder.Materials and Methods: The phenotype of BM cells that can migrate to the damaged liver was characterized by in vitro migration assay. The migrated cells were analyzed by flowcytometry (FC) and immunocytochemistry (IC). To know the potential for hepatic differentiation, these cells were cultured on extracellular matrix protein-coated plate containing 10% damaged liver serum supplemented medium. The extent of differentiation of BM cells into HEPATOCYTES was determined by analyzing hepatic genes (HNF-4a, albumin, TDO, TAT) and proteins (albumin and CK 18) expression, and also by functional assay (CYP450 activity). Therapeutic potential of BM-derived cells was tested in HA mice model. The liver of male HA mice was perturbed with acetaminophen to facilitate the engraftment and differentiation of lineage-depleted (Lin-) enhanced green fluorescent protein (eGFP)- expressing female BMCs. The fusion, if any, between donor and recipient cells was identified by quantitative polymerase chain reaction (PCR) and fluorescent in situ hybridization (FISH) assays. The donor-derived HEPATOCYTES expressing FVIII was determined by immunohistochemistry (IHC). This was confirmed by reverse transcriptase (RT)-PCR analysis of the light chain FVIII gene. The phenotypic correction in recipient HA mice was assessed by ELISA, Chromogenix and activated partial thromboplastin time (aPTT) based assays of plasma FVIII levels, and tail-clip challenge.Results: In vitro studies showed that the damaged liver tissue is capable of inducing the migration of a distinct population of BM cells, phenotypically characterized as Lin-CXCR4+OSMRb+. The competent cells of the BM can differentiate into albumin and cytoketarin-18 expressing HEPATOCYTES, which were functionally active as resorufin was produced from 5-pentoxyresorufin. In the recipient HA mice, BM-derived cells expressed the markers of both HEPATOCYTES (albumin and cytokeratin-18) and endothelial cells (von Willbrand factor). The results of quantitative PCR and FISH followed by IC suggested that hepatocyte obtained from BM-derived cells was due to direct differentiation and not due to cell fusion. The recipient HA mice expressed FVIII light chain gene and protein. The coagulation assay confirmed that the plasma FVIII activity is maintained at 20.4±3.6% of normal pooled plasma activity for more than a year without forming its inhibitor. Furthermore, 80% of the test mice survived in a tail-clip challenge during the study.Conclusion: This report demonstrated that BM cells rescued the bleeding phenotype in HA mice, suggesting a potential therapy for this and other related disorders.

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